PROJECT SUMMARY We hypothesize that Protein Kinase C (PKC) agonists such as the non-tumorigenic Bryoids combined with HDAC inhibitors (HDACi) represent a valuable pharmacological approach to purge latent HIV-1 from cellular reservoirs. We also hypothesize that a combination of a Bryoid and an HDACi co-encapsulated in a long- circulation pegylated immunonanosomes coated with anti-PD-L1 nanobodies will provide efficient HIV latency activation and immunological depletion of latent reservoirs. This combination nanosomal approach has the added benefit of concomitantly and significantly reducing systemic toxicities of both PKC and HDACi. Our Phase I Specific Aims are: (1) construct and characterize nanosomes containing the Bryoid, Bryostatin-1 in the lipid membrane and nanosomes of HDAC inhibitors SAHA, Romidepsin and Panobinostat in the aqueous core; (2) evaluate the cytotoxicity of Bryostatin-1, HDACi and of their corresponding nanosomes in cell culture models, and in vivo cytotoxicity and biodistribution in BALB/c mouse model to select most efficacious combination; and (3) evaluate the synergistic and anti-HIV-1 latency antagonistic effects of two nanosomes of different combination of Bryostatin-1 and the alternate HDAC inhibitors in cell culture and in humanized mice models of HIV latency. We plan to conduct this Proof-of-Concept study over a 24-month period with a multidisciplinary team of 1.10 full-time equivalents (FTE) of an engineer, virologist, and manufacturing technician. Additionally, our lean but experienced research team will be advised and supported by Dr. Robert F. Siliciano, Professor of Medicine, Johns Hopkins University School of Medicine and Investigator, Howard Hughes Medical Institute who will also provide support with respect to the ex vivo evaluation of the combination PKC-HDACi nanosomes; Dr. Eduardo Munoz, University of Cordoba, Spain, an immunologist, an HIV latency researcher and Scientific Advisor to Aphios Corporation; Dr. Santiago Moreno, Head, Infectious Diseases, Ramn y Cajal Hospital and Professor of Infectious Diseases, University of Alcal, Madrid, Spain, an HIV clinician and Principal Investigator of an ongoing Phase I/IIa clinical trial of Bryostatin-1 for HIV latency in HIV patients on cART; and Dr. Joseph L. Bryant, D.V.M at the Institute of Human Virology, University of Maryland Medical School, an HIV animal model expert, who will lead the in vivo toxicity and efficacy animal studies planned. We will establish a Scientific Advisory Review Panel with Drs. Siliciano, Bryant, Munoz and Santiago for the research. Should we be successful in achieving our Phase I milestones, we will prepare and propose a comprehensive Phase II research and development program which will: (1) systematically construct immunonanosomes by coating the most promising nanosomes identified in Phase I anti-PD-L1 nanobodies, and physically, chemically and biologically characterize the immunonanosomes; (2) evaluate most potent HIV latency drug combination in an induction/activation therapy protocol in the SHIV/macaque or alternative model of viral persistence and latency; and (3) determine the potential effects of treatment on selected immunological functions during induction therapy. The end-goal of our research program is to develop a combination therapeutic to clear and free HIV patients from latent viruses, curing a chronic disease and reducing the burden of personal toxicities and ending the economic burden of this disease in both developing and developed countries. In order to reach these objectives, we plan to conduct rigorous clinical studies in a Phase III clinical research program with a commercial partner/investor in which we will: (1) perform cGMP manufacturing of HIV latency combination therapeutic at the pilot-scale level; (2) establish a Drug Master File; (3) design IND-enabling preclinical studies and Phase I/II clinical trials; (4) prepare pre-IND package; and (5) establish and conduct pre-IND meeting with FDA and file an IND with the FDA. We will then conduct Phase I and II clinical trials, and license the therapeutic to a multinational pharmaceutical company such as Roche, J&J, Pfizer, Merck or GSK. In summary, we will utilize Aphios? proprietary critical fluid nanosomes (CFN) process for the formation of small, uniform liposomes for the co-encapsulation of Bryoids and HDAC inhibitors [US Patent, Castor, 2014], and take advantage of promising data we have generated from preliminary studies [Perez et al., 2010], to develop responsive protocols for Phase I and II clinical studies in HIV-1 patients being treated with cART and having a suppressed viral load [Gutirrez et al., 2016]. The outcomes of the proposed studies will inform product development for HIV latency, and fast-track the field to complete elimination of HIV infection and sterilizing cure.